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Nucleotide sequence of ornithine decarboxylase antizyme cDNA from Xenopus laevis
ilu
BB, Biochi~& ELSEVIER
Biochimica et Biophysica Acta 1262 (1995) 83-86
et BiophysicaAfta
Short Sequence-Paper
Nucleotide sequence of ornithine decarboxylase antizyme cDNA from Xenopus laevis Tamotsu Ichiba, Senya Matsufuji, Youichi Miyazaki, Shin-ichi Hayashi
*
Department of Nutrition, The Jikei University School of Medicine, Minato-ku, Tokyo 105, Japan Received 7 February 1995; accepted 6 March 1995
Abstract
An ornithine decarboxylase antizyme cDNA was obtained from Xenopus laevis liver and its sequence was determined. The cDNA consists of two major open reading frames as found in mammalian antizymes, which require + 1 ribosomal frameshifting for its translation. Sequences important for frameshifting, namely the frameshift site and downstream stimulatory pseudoknot determined in the rat mRNA, are conserved. Keywords: Ornithine decarboxylase antizyme; Frameshift; cDNA; (X. laevis)
Omithine decarboxylase (ODC), a key enzyme of polyamine biosynthesis, is dramatically induced by various stimuli [1] and rapidly degraded upon accumulation of intracellular polyamines, which is now known to be mediated by ODC antizyme [2,3]. ODC antizyme is a polyamine-inducible ODC-inhibitory protein which binds with the enzyme at high affinity [4-6]. We previously cloned cDNA [7] and gene [8] of rat antizyme and demonstrated that antizyme accelerates degradation of ODC in both in vivo [9] and in vitro [10] systems. We also demonstrated that the proteinase responsible for antizymedependent and ubiquitin-independent ODC degradation is the 26S proteasome, which had been thought to degrade mostly ubiquitinated proteins [11]. Recently, we and others showed that antizyme represses polyamine uptake [12,13]. Thus, antizyme negatively regulates cellular polyamine contents in at least three different ways, namely inhibition of ODC activity, acceleration of its degradation, and inhibition of polyamine uptake. Another characteristic feature of rat antizyme is its translational regulation by polyamines [14]. Rat antizyme mRNA has two major open reading frames (ORFs) [8,14]. The functional domains are encoded by the larger ORF
'~ The nucleotide sequence data reported in this paper have been submitted to the E M B L / G e n B a n k / D D B J Data Libraries under the accession number D32141. * Corresponding author. Fax: + 81 3 34351922. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 0 6 2 - 3
(ORF2) which lacks initiator codons and overlaps to the initiating frame (ORF 1) [ 15,16]. Polyamine-dependent + 1 frameshifting at the last codon of ORF1 is needed for antizyme translation [ 14,17]. This is the only known example of vertebrate cellular gene expressed through programmed frameshifting, which is used rather widely in viral gene expression [18]. Antizyme mRNA from human also requires frameshifting for its expression [14,19]. Antizymes homologous to that in mammalian cells seem to be present in lower vertebrates. Polyamine-inducible ODC-inhibitory protein has been found in chicken liver [20] and primary-cultured hepatocytes of Xenopus laevis [21]. Messenger RNAs hybridizable with rat antizyme cDNA were also detected in avian, frog and fish livers [7]. Antizymes from a plant [22] and Escherichia coli [23] have also been reported: the nucleotide sequence of the latter shows no homology with the rat mRNA. In the present study, we report the sequence of Xenopus antizyme cDNA, showing that translational frameshifting is also necessary for its expression. To amplify the Xenopus cDNA by PCR, we first used rat antizyme cDNA primers. The amplified DNA fragment (about 550 bp) was gel purified and sequenced directly. Sequences of 5' and 3' regions were obtained with RACE (Rapid Amplification of cDNA Ends) [24] and four clones of each region were sequenced. Xenopus antizyme cDNA is 997 bp in length (Fig. 1), which shows 65% identity to the rat cDNA at the nucleotide level. As seen in the rat cDNA, there are two
T. lchiba et al. / Biochimica et Biophysica Acta 1262 (1995) 8 3 - 8 6
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GGAGATTGTCCGACGGACGCGACAGAAAGCGGGGGATTIq~ ORFI
ORFI ORF2
TGGTTCGGAGAC C A G A G A ~ C G ~ T G A A A T C M V K
TACTC.~TAGC CATI~C 'i "lu GCC A G A G ~ G ~ A C - G A . , ~ T - a ~ . ~ G A ~ C L N S H C F A R E K E G N K R N
C TC CCTCCAACGGA 90 S L 0 R I 9
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GAC~ @ C ~ ~ ~ A G TATACC CAGTAGT A G ~ 180 D A M P L L S I P $ $ S E S 39 V Y P V V V K
ORFI ORF2
G'Pi~ CAGGGC"PII2'I"P~ ~ , ~ ' I I 3 T A G T . , ~ C C'I~C~'II2C CC.~GCC"B~GG~3G TG~ ~ S R A S F N C C S N L G P G P R W ~ $ V P G L L S I A V V T W V P G L G G V P~
ORF2
GG~GAGGGAATAGTCAGAGGGATCACAATCTTI~AGCTAATTTA~q`i.ATTCTGATAATAGGCTGAATATAACAGAGGAGCTTACA $ G R G N $ Q R D H N L S A N L F Y S D N R L N I T E E L T
360 98
TCCAACAACAGGACCAGAATAC T C A A T G T C C A A T C A A G T C T G A C A G A T G G C A A G C A A G T G A G ~ A G A G C T G T G C S N N R T R I L N V O S S L T D G K O V $ W R A V
450 128
V
C C ~ ACC CACCCC ~ G
A ~ C CA 2 7 0
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TAAACAATAACAAC I~ N N N N
CTCTACATAGAAATCCCAAGTC43TACTC TACC TGA~AGCAAAGACAG'i'I'i'iGCAATTC T A C T G G A A T A T G C A G A A G A G C A A C T I U A A L Y I E I P S G T L P D G S K D S F A I L L E Y A E E 0 L 0
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GTGGATCATGTCTTTATCTGCTTCCACAAGAACAGC43ATGATAGAGCTATC42
630 188
V
D
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ATACC TGGACATCCCC q'I~TCC C TAAGAGAC CAGATGC ~ I P G H P L V P K R P D A C
R
A
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TTC TCCGAAC'iq q C A G A T T C ~ C T I Z ~ A G A T T G T G L
L
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TTTATC43C TTACACC TIq~AAAGAGATTC TTC T G A T G A A G A ~ - ~ C F M A Y T F E R D S $ D E D
V
A 720 216
GCCC 2 F g S A A ~ ~'i'i'±'r PTC~CCTCCCCACATC TCGAGCTGGAATGTGAGAAATCATGTCAATTTAAGCTGTI~A 'ITI'ATGC TTGTGCCCATA~TCTGAGAGAAGACACAAGA'I'I'I TGAGA'ITI'IX2ATCATATTC TGAA'i'i'ri'i'rrITIGTCTCgSACGTTAT GAATAAATGCCACqL~CAATITATIC-4~TATATGAATGGAGGTTTAATGTGTTITGTGATAGTAC TTITCTTTCATTCTAAATAAAACATTT CTATTTT 997
810 900 990
Fig. 1. Nucleotide and deduced amino acid sequences of ODC antizyme from Xenopus laevis. Two potential initiator codons are circled and the termination codons are boxed. Amino acid sequence predicted after + 1 frameshifting at the same site as rat is underlined.
major ORFs in Xenopus antizyme (Fig. 1). Two potential initiator codons of ORF1, as well as the shift site sequence, TCCTGAT (where the terminator of ORF1 is underlined) of the rat cDNA [14] are totally conserved. Plus 1 frameshifting at the same site as rat mRNA will make a 216 amino acids protein with a molecular mass of 24 kDa, or an 189 amino acids protein with a molecular mass of 21 kDa, depending on the use of first or second initiator. The deduced amino acid sequence translated from the first initiator holds 74% identity to rat antizyme (Fig. 3). The 649 bp potential coding region shows 73% of nucleotide identity to the rat sequence. The 65 bp 5'-untranslated region (UTR) and 283 bp 3'-UTR exhibit less identity to the rat sequence, 59% and 49%, respectively.
Human Rat
Xeno19us
Compared with the rat and human sequence, there are only five nucleotides different within nucleotides 225-303 (Fig. 2). It was demonstrated that the corresponding region of rat mRNA contains all the essential components for the frameshifting, namely the shift site, terminator of ORF1 and a downstream pseudoknot, a tertiary structure of mRNA known as a stimulator of virus frameshifting [25,26]. It was also shown that the region itself can direct polyamine-dependent frameshifting when inserted between reporter genes [14]. Among these five nucleotide changes, a C ~ T change immediately 5' to the shift site did not affect frameshift efficiency in reticulocyte lysate [14]. The other four are within the pseudoknot, but not involved in the base pairing for pseudoknot (Fig. 2). Including this
190 C C T C G G T G G T G C T C ~ C C C C T C A C C C A C C C C T G A A G A T C C C A G G T G C I ~ G A G G G A A T A G T C A G A G G G A T C A C A A T 190 C C T C G G T C 4 3 T G C T C ~ G T C C C T C A C C C A C ~ C C T G A A G A T C C C A G G ~ C G A G G G A A C A G T C A G C G C 4 ~ A T C A C A G T 225 C C T C G G T G G T G T T C ~ T C C C T C A C C C A C C C C T G A A G A T C C C A G G ~ G A G G G A A T A G T C A G A G G G A T C A C A A T
268 268 303
Fig. 2. Partial sequence comparison of human, rat and Xenopus antizymes. Nucleotide 2 2 5 - 3 0 3 of the Xenopus sequence and the corresponding human and rat sequence are shown. The termination codons of ORF1 are boxed. Stems of the pseudoknot structure are underlined. The identical residues are shown as asterisk.
T. lchiba et a l . / Biochimica et Biophysica Acta 1262 (1995) 83-86
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I:MVKSSLQRILNSHCFAREKEGDKPSATIHASRTMPLLSLHSRGGSSSESSRVSLHCCSNP I:MVKSSLQRILNSHCFAREKEGDKRSATLHASRTMPLLSQHSRGGCSSESSRVALHCCSNL I:MVKSSLQRILNSHCFAREKEGNKRND---A---MPLLSI .... PSSSESSRASFNCCSNL
61:GPGPRWC~DAPHPPLKIPGGRGNSQRDHNLSANLFYSDDRLNVTEELTSNDKTRILNVQS 61:GPGPRWCS~VPHPPLKIPGGRGNSQRDHSLSASILYSDERLNVTEEPTSNDKTRVLSIQC 51:GPGPRWC~DVPHPPLKIPGGRGNSQRDHNLSANLFYSDNRLNITEELTSNNRTRILNVQS 121:RLTDAKRINWRTVLSGGSLYIEIPGGALPEGSKDSFAVLLEFAEEQLRADHVFICFHKNR 121:TLTEAKQVTWRAVWNGGGLYIELPAGPLPEGSKDSFAALLEFAEEQLRADHVFICFPKNR III:SLTDGKQVSWRAVLNNNNLYIEIPSGTLPDGSKDSFAILLEYAEEQLQVDHVFICFHKNR
181:EDRAALLRTFSFLGFEIVRPGHPLVPKRPDACFMAYTFERESSGEEEE 181:EDRAALLRTFSFLGFEIVRPGHPLVPKRPDACFMVYTLEREDPGEED171:DDRAMLLRTFRFLGFEIVIPGHPLVPKRPDACFMAYTFERDSSD-ED*** * * * * * ******* * * * * * * * * * * * * * * * ** ** . Fig. 3. Alignment of deduced amino acid sequences of human, rat and Xenopus antizyme. The border of ORFI- and ORF2-coded sequence is shown by a vertical line. The PEST sequences are underlined. The identical residues are shown as asterisks.
region, nucleotides 199-313 is the most conserved segment between rat and Xenopus mRNA ( 1 1 0 / 1 1 5 identity). These findings strongly suggest that the + 1 frameshifting at the end of ORF1 also takes place for Xenopus antizyme mRNA, and that the downstream pseudoknot is an important element for this process. We and others previously examined the effects of a series of deletion mutants of rat antizyme [15,16]. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for binding to ODC and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization. Unexpectedly, sequences which are necessary for binding, inhibition, and destabilization are less conserved between Xenopus and rat (Fig. 3). The reason for the discrepancy is unknown. It should be noted that sequences which are necessary for the inhibition of polyamine transport are not determined in the present time. Rat antizyme itself is a short-lived protein [5] and contains a PEST sequence which is commonly observed in rapidly turning-over proteins [27]. Rat PEST sequence, located at residues 100-112, was not conserved in human and Xenopus antizyme. Xenopus antizyme contains, however, another PEST sequence situated at residues 24-41 (PEST score is 0.023, respectively). The meaning of the PEST sequence of antizyme is obscure. In summary, our present data show that sequences which are necessary for frameshifting in rat antizyme are highly conserved in Xenopus antizyme, suggesting that ribosomal frameshifting occurs in the translation of Xenopus antizyme mRNA.
We thank Drs. Y. Murakami and M. Nishiyama for technical advices and encouragement.
References [1] Pegg, A.E. (1988) Cancer Res. 48, 759-774. [2] Kanamoto, R., Kameji, T., Iwashita, S., Igarashi, K. and Hayashi, S. (1993) J. Biol. Chem. 268, 9393-9399. [3] Hayashi, S. and Murakami, Y. (1995) Biochem. J. 306, 1-10. [4] Fong, W.F., Heller, J.S. and Canellakis, E.S. (1976) Biochim. Biophys. Acta 428, 456-465. [5] Heller, J.S., Fong, W.F. and Canellakis, E.S. (1976) Proc. Natl. Acad. Sci. USA 73, 1858-1862. [6] Kitani, T. and Fujisawa, H. (1984) J. Biol. Chem. 259, 10036-10040. [7] Matsufuji, S., Miyazaki, Y., Kanamoto, R., Kameji, T., Murakami, Y., Baby, T.G., Fujita, K., Ohno, T. and Hayashi, S. (1990) J. Biochem. Tokyo 108, 365-371. [8] Miyazaki, Y., Matsufuji, S. and Hayashi, S. (1992) Gene 113, 191-197. [9] Murakami, Y., Matsufuji, S., Miyazaki, Y. and Hayashi, S. (1992) J. Biol. Chem. 267, 13138-13141. [10] Murakami, Y., Tanaka, K., Matsufuji, S., Miyazaki, Y. and Hayashi, S. (1992) Biochem. J. 283, 661-664. [111 Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K. and Ichihara, A. (1992) Nature 360, 597599. [12] Mitchell, J.L., Judd, G.G., Bareyal-Leyser, A. and Ling, S.Y, (1994) Biochem. J. 299, 19-22. [13] Suzuki, T., He, Y., Kashiwagi, K., Murakami, Y., Hayashi, S. and Igarashi, K. (1994) Proc. Natl. Acad. Sci. USA 91, 8930-8934. [14] Matsufuji, S., Matsufuji, T., Miyazaki, Y., Murakami, Y., Atkins, J.F., Gesteland, R.F, and Hayashi, S. (1995) Cell 80, 51-60. [15] Ichiba, T., Matsufuji, S., Miyazaki, Y., Murakami, Y., Tanaka, K., Ichihara, A., and Hayashi, S. (1994) Biochem. Biophys. Res. Commun. 200, 1721-1727. [16] Li, X. and Coffino, P. (1994) Mol. Cell Biol. 14, 87-92.
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[17] Gesteland, R.F., Weiss, R.B., and Atkins, J.F. (1992) Science 257, 1640-1641. [18] Atkins, J.F., Weiss, R.B. and Gesteland, R.F. (1990) Cell 62, 413-423. [19] Tewari, D.S., Qian, Y., Thornton, R.D., Pieringer, J., Taub, R., Mochan, E. and Tewari, M. (1994) Biochim. Biophys. Acta 1209, 293-295. [20] Grillo, M.A., Bedino, S. and Testore, G. (1980) Int. J. Biochem. 11, 37-42. [2l] Baby, T.G. and Hayashi, S. (1991) Biochim. Biophys. Acta 1092, 161-164. [22] Koromilas, A.E. and Kyriakidis, D.A. (1987) Plant Growth Regul. 6, 267-275.
[23] Canellakis, E.S., Paterakis, A.A., Huang, S.C., Panagiotidis, C.A. and Kyriakidis, D.A. (1993) Proc. Natl. Acad. Sci. USA 90, 71297133. [24] Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002. [25] Jacks, T., Madhani, H.D., Masiarz, F.R. and Varmus, H.E. (1988) Cell 55, 447-458. [26] Brierley, I., Digard, P. and Inglis, S.C. (1989) Cell 57, 537-547. [27] Rogers, S., Wells, R. and Rechsteiner, M. (1986) Science 234, 364-368.
BB, Biochi~& ELSEVIER
Biochimica et Biophysica Acta 1262 (1995) 83-86
et BiophysicaAfta
Short Sequence-Paper
Nucleotide sequence of ornithine decarboxylase antizyme cDNA from Xenopus laevis Tamotsu Ichiba, Senya Matsufuji, Youichi Miyazaki, Shin-ichi Hayashi
*
Department of Nutrition, The Jikei University School of Medicine, Minato-ku, Tokyo 105, Japan Received 7 February 1995; accepted 6 March 1995
Abstract
An ornithine decarboxylase antizyme cDNA was obtained from Xenopus laevis liver and its sequence was determined. The cDNA consists of two major open reading frames as found in mammalian antizymes, which require + 1 ribosomal frameshifting for its translation. Sequences important for frameshifting, namely the frameshift site and downstream stimulatory pseudoknot determined in the rat mRNA, are conserved. Keywords: Ornithine decarboxylase antizyme; Frameshift; cDNA; (X. laevis)
Omithine decarboxylase (ODC), a key enzyme of polyamine biosynthesis, is dramatically induced by various stimuli [1] and rapidly degraded upon accumulation of intracellular polyamines, which is now known to be mediated by ODC antizyme [2,3]. ODC antizyme is a polyamine-inducible ODC-inhibitory protein which binds with the enzyme at high affinity [4-6]. We previously cloned cDNA [7] and gene [8] of rat antizyme and demonstrated that antizyme accelerates degradation of ODC in both in vivo [9] and in vitro [10] systems. We also demonstrated that the proteinase responsible for antizymedependent and ubiquitin-independent ODC degradation is the 26S proteasome, which had been thought to degrade mostly ubiquitinated proteins [11]. Recently, we and others showed that antizyme represses polyamine uptake [12,13]. Thus, antizyme negatively regulates cellular polyamine contents in at least three different ways, namely inhibition of ODC activity, acceleration of its degradation, and inhibition of polyamine uptake. Another characteristic feature of rat antizyme is its translational regulation by polyamines [14]. Rat antizyme mRNA has two major open reading frames (ORFs) [8,14]. The functional domains are encoded by the larger ORF
'~ The nucleotide sequence data reported in this paper have been submitted to the E M B L / G e n B a n k / D D B J Data Libraries under the accession number D32141. * Corresponding author. Fax: + 81 3 34351922. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 0 6 2 - 3
(ORF2) which lacks initiator codons and overlaps to the initiating frame (ORF 1) [ 15,16]. Polyamine-dependent + 1 frameshifting at the last codon of ORF1 is needed for antizyme translation [ 14,17]. This is the only known example of vertebrate cellular gene expressed through programmed frameshifting, which is used rather widely in viral gene expression [18]. Antizyme mRNA from human also requires frameshifting for its expression [14,19]. Antizymes homologous to that in mammalian cells seem to be present in lower vertebrates. Polyamine-inducible ODC-inhibitory protein has been found in chicken liver [20] and primary-cultured hepatocytes of Xenopus laevis [21]. Messenger RNAs hybridizable with rat antizyme cDNA were also detected in avian, frog and fish livers [7]. Antizymes from a plant [22] and Escherichia coli [23] have also been reported: the nucleotide sequence of the latter shows no homology with the rat mRNA. In the present study, we report the sequence of Xenopus antizyme cDNA, showing that translational frameshifting is also necessary for its expression. To amplify the Xenopus cDNA by PCR, we first used rat antizyme cDNA primers. The amplified DNA fragment (about 550 bp) was gel purified and sequenced directly. Sequences of 5' and 3' regions were obtained with RACE (Rapid Amplification of cDNA Ends) [24] and four clones of each region were sequenced. Xenopus antizyme cDNA is 997 bp in length (Fig. 1), which shows 65% identity to the rat cDNA at the nucleotide level. As seen in the rat cDNA, there are two
T. lchiba et al. / Biochimica et Biophysica Acta 1262 (1995) 8 3 - 8 6
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GGAGATTGTCCGACGGACGCGACAGAAAGCGGGGGATTIq~ ORFI
ORFI ORF2
TGGTTCGGAGAC C A G A G A ~ C G ~ T G A A A T C M V K
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C TC CCTCCAACGGA 90 S L 0 R I 9
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360 98
TCCAACAACAGGACCAGAATAC T C A A T G T C C A A T C A A G T C T G A C A G A T G G C A A G C A A G T G A G ~ A G A G C T G T G C S N N R T R I L N V O S S L T D G K O V $ W R A V
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A ~ C CA 2 7 0
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CTCTACATAGAAATCCCAAGTC43TACTC TACC TGA~AGCAAAGACAG'i'I'i'iGCAATTC T A C T G G A A T A T G C A G A A G A G C A A C T I U A A L Y I E I P S G T L P D G S K D S F A I L L E Y A E E 0 L 0
540 158
GTGGATCATGTCTTTATCTGCTTCCACAAGAACAGC43ATGATAGAGCTATC42
630 188
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TTTATC43C TTACACC TIq~AAAGAGATTC TTC T G A T G A A G A ~ - ~ C F M A Y T F E R D S $ D E D
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GCCC 2 F g S A A ~ ~'i'i'±'r PTC~CCTCCCCACATC TCGAGCTGGAATGTGAGAAATCATGTCAATTTAAGCTGTI~A 'ITI'ATGC TTGTGCCCATA~TCTGAGAGAAGACACAAGA'I'I'I TGAGA'ITI'IX2ATCATATTC TGAA'i'i'ri'i'rrITIGTCTCgSACGTTAT GAATAAATGCCACqL~CAATITATIC-4~TATATGAATGGAGGTTTAATGTGTTITGTGATAGTAC TTITCTTTCATTCTAAATAAAACATTT CTATTTT 997
810 900 990
Fig. 1. Nucleotide and deduced amino acid sequences of ODC antizyme from Xenopus laevis. Two potential initiator codons are circled and the termination codons are boxed. Amino acid sequence predicted after + 1 frameshifting at the same site as rat is underlined.
major ORFs in Xenopus antizyme (Fig. 1). Two potential initiator codons of ORF1, as well as the shift site sequence, TCCTGAT (where the terminator of ORF1 is underlined) of the rat cDNA [14] are totally conserved. Plus 1 frameshifting at the same site as rat mRNA will make a 216 amino acids protein with a molecular mass of 24 kDa, or an 189 amino acids protein with a molecular mass of 21 kDa, depending on the use of first or second initiator. The deduced amino acid sequence translated from the first initiator holds 74% identity to rat antizyme (Fig. 3). The 649 bp potential coding region shows 73% of nucleotide identity to the rat sequence. The 65 bp 5'-untranslated region (UTR) and 283 bp 3'-UTR exhibit less identity to the rat sequence, 59% and 49%, respectively.
Human Rat
Xeno19us
Compared with the rat and human sequence, there are only five nucleotides different within nucleotides 225-303 (Fig. 2). It was demonstrated that the corresponding region of rat mRNA contains all the essential components for the frameshifting, namely the shift site, terminator of ORF1 and a downstream pseudoknot, a tertiary structure of mRNA known as a stimulator of virus frameshifting [25,26]. It was also shown that the region itself can direct polyamine-dependent frameshifting when inserted between reporter genes [14]. Among these five nucleotide changes, a C ~ T change immediately 5' to the shift site did not affect frameshift efficiency in reticulocyte lysate [14]. The other four are within the pseudoknot, but not involved in the base pairing for pseudoknot (Fig. 2). Including this
190 C C T C G G T G G T G C T C ~ C C C C T C A C C C A C C C C T G A A G A T C C C A G G T G C I ~ G A G G G A A T A G T C A G A G G G A T C A C A A T 190 C C T C G G T C 4 3 T G C T C ~ G T C C C T C A C C C A C ~ C C T G A A G A T C C C A G G ~ C G A G G G A A C A G T C A G C G C 4 ~ A T C A C A G T 225 C C T C G G T G G T G T T C ~ T C C C T C A C C C A C C C C T G A A G A T C C C A G G ~ G A G G G A A T A G T C A G A G G G A T C A C A A T
268 268 303
Fig. 2. Partial sequence comparison of human, rat and Xenopus antizymes. Nucleotide 2 2 5 - 3 0 3 of the Xenopus sequence and the corresponding human and rat sequence are shown. The termination codons of ORF1 are boxed. Stems of the pseudoknot structure are underlined. The identical residues are shown as asterisk.
T. lchiba et a l . / Biochimica et Biophysica Acta 1262 (1995) 83-86
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I:MVKSSLQRILNSHCFAREKEGDKPSATIHASRTMPLLSLHSRGGSSSESSRVSLHCCSNP I:MVKSSLQRILNSHCFAREKEGDKRSATLHASRTMPLLSQHSRGGCSSESSRVALHCCSNL I:MVKSSLQRILNSHCFAREKEGNKRND---A---MPLLSI .... PSSSESSRASFNCCSNL
61:GPGPRWC~DAPHPPLKIPGGRGNSQRDHNLSANLFYSDDRLNVTEELTSNDKTRILNVQS 61:GPGPRWCS~VPHPPLKIPGGRGNSQRDHSLSASILYSDERLNVTEEPTSNDKTRVLSIQC 51:GPGPRWC~DVPHPPLKIPGGRGNSQRDHNLSANLFYSDNRLNITEELTSNNRTRILNVQS 121:RLTDAKRINWRTVLSGGSLYIEIPGGALPEGSKDSFAVLLEFAEEQLRADHVFICFHKNR 121:TLTEAKQVTWRAVWNGGGLYIELPAGPLPEGSKDSFAALLEFAEEQLRADHVFICFPKNR III:SLTDGKQVSWRAVLNNNNLYIEIPSGTLPDGSKDSFAILLEYAEEQLQVDHVFICFHKNR
181:EDRAALLRTFSFLGFEIVRPGHPLVPKRPDACFMAYTFERESSGEEEE 181:EDRAALLRTFSFLGFEIVRPGHPLVPKRPDACFMVYTLEREDPGEED171:DDRAMLLRTFRFLGFEIVIPGHPLVPKRPDACFMAYTFERDSSD-ED*** * * * * * ******* * * * * * * * * * * * * * * * ** ** . Fig. 3. Alignment of deduced amino acid sequences of human, rat and Xenopus antizyme. The border of ORFI- and ORF2-coded sequence is shown by a vertical line. The PEST sequences are underlined. The identical residues are shown as asterisks.
region, nucleotides 199-313 is the most conserved segment between rat and Xenopus mRNA ( 1 1 0 / 1 1 5 identity). These findings strongly suggest that the + 1 frameshifting at the end of ORF1 also takes place for Xenopus antizyme mRNA, and that the downstream pseudoknot is an important element for this process. We and others previously examined the effects of a series of deletion mutants of rat antizyme [15,16]. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for binding to ODC and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization. Unexpectedly, sequences which are necessary for binding, inhibition, and destabilization are less conserved between Xenopus and rat (Fig. 3). The reason for the discrepancy is unknown. It should be noted that sequences which are necessary for the inhibition of polyamine transport are not determined in the present time. Rat antizyme itself is a short-lived protein [5] and contains a PEST sequence which is commonly observed in rapidly turning-over proteins [27]. Rat PEST sequence, located at residues 100-112, was not conserved in human and Xenopus antizyme. Xenopus antizyme contains, however, another PEST sequence situated at residues 24-41 (PEST score is 0.023, respectively). The meaning of the PEST sequence of antizyme is obscure. In summary, our present data show that sequences which are necessary for frameshifting in rat antizyme are highly conserved in Xenopus antizyme, suggesting that ribosomal frameshifting occurs in the translation of Xenopus antizyme mRNA.
We thank Drs. Y. Murakami and M. Nishiyama for technical advices and encouragement.
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